Soybean is the lowest-cost source of vegetable oil. Soybean oil accounts for about 70% of the 14 billion pounds of edible oil consumed in the United States and is a major edible oil worldwide. It is used in baking, frying, salad dressing, margarine, and a multitude of processed foods. Soybean is agronomically well-adapted to many parts of the U.S. In the late 1980s sixty million acres of soybean were planted annually in the U.S.
Soybean products are also a major element of foreign trade. Approximately, thirty million metric tons of soybeans, twenty-five million metric tons of soybean meal, and one billion pounds of soybean oil were exported in 1987/88. Nevertheless, increased foreign competition has lead to recent declines in soybean acreage and production in the U.S. The low cost and ready availability of soybean oil provides an excellent opportunity to upgrade this commodity oil into higher value specialty oils that add value to soybean crop for the U.S. farmer and enhance U.S. trade.
The specific functionalities and health attributes of edible oils are determined largely by their fatty acid composition. Soybean oil derived from commercial varieties is composed primarily of 11% palmitic (16:0), 4% stearic (18:0), 24% oleic (18:1), 54% linoleic (18:2) and 7% linolenic (18:3) acids. Palmitic and stearic acids are, respectively, 16- and 18-carbon-long, saturated fatty acids. Oleic, linoleic and linolenic are 18-carbon-long, unsaturated fatty acids containing one, two and three double bonds, respectively. Oleic acid is also referred to as a "monounsaturated" fatty acid, while linoleic and linolenic acids are also referred to as "polyunsaturated" fatty acids.
Soybean oil is high in saturated fatty acids when compared to other sources of vegetable oil and contains a low proportion of oleic acid relative to the total fatty acid content of the soybean seed. These characteristics do not satisfy recommendations for the consumption of fats from the American Heart Association.
Recent research efforts have examined the role that monounsaturated fatty acid plays in reducing the risk of coronary heart disease. In the past, it was believed that monounsaturates, in contrast to saturates and polyunsaturates, had no effect on serum cholesterol and coronary heart disease risk. Several recent human clinical studies suggest that diets high in monounsaturated fat may reduce the "bad" (low-density lipoprotein) cholesterol while maintaining the "good" (high-density lipoprotein) cholesterol. (See Mattson, et al., Journal of Lipid Research (1985) 26:194-202). The significance of monounsaturated fat in the diet was confirmed by international researchers from seven countries at the Second Colloquium on Monounsaturated Fats sponsored by the National Heart, Lung and Blood Institutes in 1987.
Soybean oil is also relatively high in polyunsaturated fatty acids--at levels far in excess of essential dietary requirements. These fatty acids oxidize readily to give off-flavors and reduce the performance of unprocessed soybean oil. The stability and flavor of soybean oil is improved by hydrogenation, which chemically reduces the double bonds. However, this processing reduces the economic attractiveness of soybean oil.
A soybean oil low in total saturates and polyunsaturates and high in monounsaturate would provide significant health benefits to human consumers as well as economic benefit to oil processors. Such soybean varieties will also produce valuable meal for use as animal feed.
Another type of differentiated soybean oil is an edible fat for confectionery uses. More than two billion pounds of cocoa butter, the most expensive edible oil, are produced worldwide. The U.S. imports several hundred million dollars worth of cocoa butter annually. The high and volatile prices and uncertain supply of cocoa butter have encouraged the development of cocoa butter substitutes. The fatty acid composition of cocoa butter is 26% palmitic, 34% stearic, 35% oleic and 3% linoleic acids. Cocoa butter's unique fatty acid composition and distribution on the triglyceride molecule confer on it properties eminently suitable for confectionery end-uses: it is brittle below 27.degree. C. and depending on its crystalline state, melts sharply at 25-30.degree. C. or 35.degree.-36.degree. C. Consequently, it is hard and non-greasy at ordinary temperatures and melts very sharply in the mouth. It is also extremely resistant to rancidity. For these reasons, a soybean oil with increased levels of palmitic and stearic acids, especially in soybean lines containing reduced levels of unsaturated fatty acids, is expected to provide a cocoa butter substitute in soybean. This will add value to oil and food processors as well as reduce the foreign import of certain tropical oils.
Only recently have serious efforts been made to improve the quality of soybean oil through plant breeding, especially mutagenesis. A wide range of fatty acid compositions have been discovered in experimental lines of soybean (Table 1). Findings from work on various oil crops suggest that the fatty acid composition of soybean oil can be significantly altered without affecting the agronomic performance of a soybean plant. However, there is no soybean mutant line with levels of saturates less than those present in commercial canola, the major competitor to soybean oil as a "healthy" oil.
TABLE 1 ______________________________________ Range of Fatty Acid Percentages Produced by Soybean Mutants Fatty Acids Range of % ______________________________________ Palmitic Acid 6-28 Stearic Acid 3-30 Oleic Acid 17-50 Linoleic Acid 35-60 Linolenic Acid 3-12 ______________________________________
There are serious drawbacks to using mutagenesis to alter fatty acid composition. It is unlikely to discover mutations a) that result in a dominant ("gain-of-function") phenotype, b) in genes that are essential for plant growth, and c) in an enzyme that is not rate-limiting and that is encoded by more than one gene. Even when some of the desired mutations are available in soybean mutant lines their introgression into elite lines by traditional breeding techniques will be slow and expensive, since the desired oil compositions in soybean are most likely to involve several recessive genes.
Recent molecular and cellular biology techniques offer the potential for overcoming some of the limitations of the mutagenesis approach, including the need for extensive breeding. Particularly useful technologies are: a) seed-specific expression of foreign genes in transgenic plants (see Goldberg et al., (1989) Cell 56:149-160), b) use of antisense RNA to inhibit plant target genes in a dominant and tissue-specific manner (see van der Krol et al., (1988) Gene 72:45-50), c) use of homologous transgenes to suppress native gene expression (see Napoli et al., (1990) The Plant Cell 2:279-289; van der Krol et al., (1990) The Plant Cell 2:291-299; Smith et al., (1990) Mol. Gen. Genetics 224:477-481), d) transfer of foreign genes into elite commercial varieties of commercial oilcrops, such as soybean (Chee et al. (1989) Plant Physiol. 91:1212-1218; Christou et al., (1989) Proc. Natl. Acad. Sci. U.S.A. 86:7500-7504; Hinchee et al., (1988) Bio/Technology 6:915-922; EPO publication 0 301 749 A2), rapeseed (De Block et al., (1989) Plant Physiol. 91:694-701!, and sunflower (Everett et al., ,(1987) Bio/Technology 5:1201 -1204), and e) use of genes as restriction fragment length polymorphism (RFLP) markers in a breeding program, which makes introgression of recessive traits into elite lines rapid and less expensive (Tanksley et al. (1989) Bio/Technology 7:257-264). However, each of these technologies requires identification and isolation of commercially-important genes.
Oil biosynthesis in plants has been fairly well-studied (see Harwood (1989) in Critical Reviews in Plant Sciences, Vol. 8 (1):1-43). The biosynthesis of palmitic, stearic and oleic acids occurs in the plastids of plant cells by the interplay of three key enzymes of the "ACP track": palmitoyl-ACP elongase, stearoyl-ACP desaturase and acyl-ACP thioesterase.
Of these three enzymes, acyl-ACP thioesterase removes the acyl chain from the carrier protein (ACP) and thus from the metabolic pathway. The same enzyme, with slightly differing efficiency, catalyzes the hydrolysis of the palmitoyl, stearoyl and oleoyl-ACP thioesters. This multiple activity leads to substrate competition between enzymes and it is the competition of acyl-ACP thioesterase and palmitoyl-ACP elongase for the same substrate and of acyl-ACP thioesterase and stearoyl-ACP desaturase for the same substrate that leads to the production of a particular ratio of palmitic, stearic and oleic acids.
Once removed from the ACP track by the action of acyl-ACP thioesterase, fatty acids are exported to the cytoplasm and there used to synthesize acyl-coenzyme A (CoA). These acyl-CoA's are the acyl donors for at least three different glycerol acylating enzymes (glycerol-3-P acyltransferase, 1 -acyl-glycerol-3-P acyltransferase and diacylglycerol acyltransferase) which incorporate the acyl moieties into triacylglycerides during oil biosynthesis.
These acyltransferases show a strong, but not absolute, preference for incorporating saturated fatty acids at positions 1 and 3 and monounsaturated fatty acid at position 2 of the triglyceride. Thus, altering the fatty acid composition of the acyl pool will drive by mass action a corresponding change in the fatty acid composition of the oil. Furthermore, there is experimental evidence that, because of this specificity, given the correct composition of fatty acids, plants can produce cocoa butter substitutes (Bafor et al., (1990) J. Amer. Oil Chemists Soc. 67:217-225).
Based on the above discussion, one possible means of altering the saturated fatty acid composition of soybean oil is to modulate the activity of acyl-ACP thioesterase in seed tissue. The biosynthesis of fatty acids proceed by the elongation of fatty acyl moieties attached to acyl carrier protein (ACP). Elongation is accomplished by the repetitive addition of acetyl units to the chain, and each of the resulting .beta.-keto acyl chains is reduced to the saturated equivalent prior to the next addition. When the growing chain reaches the 16- or 18-carbon length, the acyl-ACP can undergo two fates. It either becomes the substrate for the acyl-ACP desaturase, resulting in a monounsaturated fatty acid, or it is a substrate of acyl-ACP thioesterase which cleaves the acyl group from ACP and the chain is no longer a possible substrate for the desaturase. Thus, by either raising or lowering the level of thioesterase in a developing seed it should be possible to raise or lower, respectively, the amount of saturated fatty acids in seed oil. This modulation of activity could be achieved by either the over-production or suppression of production of the thioesterase. Such manipulation requires the use of a gene or fragment thereof which encodes a thioesterase.
Plant thioesterases which are monofunctional proteins that catalyze the hydrolysis of acyl-ACP thioesters are generally referred to as class I thioesterases. Class II thioesterase activities are usually found as components of multifunctional polypeptides and are exemplified by the enzymes from avian (Rogers and Kolattukudy (1984) Anal. Biochem 137:444-448) and rat (Naggert et al. (1988) J. Biol. Chem. 263:1146-1150). The relationship between these two classes of enzyme was not known, and the preferred approach would be to use a monofunctional activity. However, prior to the instant invention, limited research had been conducted toward the isolation of thioesterases, or their genes, from plants. There are no previous reports of such efforts directed toward the isolation of soy thioesterases. The partial purification of acyl-ACP thioesterase was reported from safflower seeds (McKeon et al., (1982) J. Biol. Chem. 257:12141-12147). This purification scheme was not useful for soybean, either because the thioesterases are different or because of the presence of other proteins such as the soybean seed storage proteins in seed extracts.
U.S. Pat. No. 5,147,792 issued to Perchorowicz et al. on Sep. 15, 1992, describes a method of shifting the fatty acid distribution in plastids towards shorter-chained species by using thioesterase II and acyl carrier protein. While Perchorowicz et al. teach a method for altering the fatty acid distribution in isolated plastids, they do not teach a method of producing such altered fatty acid profiles in whole plant cells. They further do not teach a method of producing sexually reproducing plants producing altered fatty acid profiles. The methods therefore do not provide a means for the large scale production of usable vegetable oils with new fatty acid compositions.
U.S. Pat. No. 5,298,421 issued to Davies et al. on Mar. 29, 1994, describes plant medium-chain preferring acyl-ACP thioesterases and related methods. The methods taught by Davies et al. produce plants with seed oil compositions with substantial amounts of fatty acids of less than 16 carbon atoms in length. These oils are different from the seed oils produced by normal, temperate oilseeds in this characteristic. The seed oil fatty acid compositions taught in the instant invention are not elevated in comparison to normal oils in their shorter (less than 16 carbon atom) fatty acids. The fatty acid profiles produced in the instant invention are different both from common temperate oilseeds and from the invention of Davies et al. in that they contain elevated levels of fully saturated fatty acids of 16 and 18 carbon atoms in length.